Cancer drug screen based on cell cycle uncoupling

ABSTRACT

Checkpoint gene-defective human cells are useful for screening potential anti-tumor agents. Potential therapeutic agents are screened for the ability to cause DNA accumulation or cell death in a checkpoint gene-defective human cell.

This application is a division of U.S. application Ser. No. 09/828,906filed Apr. 10, 2001 (now issued as U.S. Pat. No. 6,511,818), which is adivision of application Ser. No. 09/298,822 filed Apr. 26, 1999 (nowissued as U.S. Pat. No. 6,214,616); which is a division of Ser. No.08/620,340 filed Mar. 22, 1996 (now issued as U.S. Pat. No. 5,897,999).

This invention was made using U.S. government grants from the NIHCA43460, CA62924, CA35494, GM07309, and GM07184. Therefore the U.S.government retains certain rights to the invention.

BACKGROUND OF THE INVENTION

Precise coordination of the S and M phases of the eukaryotic cell cycleis critical not only for normal cell division, but also for effectivegrowth arrest under conditions of stress. When damaged, a cell mustcommunicate signals to both the mitotic and DNA synthesis machineries sothat a mitotic block is not followed by an extra S phase, or vice versa.The biochemical mechanisms regulating this coordination, termedcheckpoints, have been identified in lower eukaryotes, but are largelyunknown in mammalian cells¹⁻³.

DNA-damaging agents are used in the clinic to preferentially kill cancercells. However, there is a need in the art to discover additionaltherapeutic agents which are selectively toxic to cancer cells.

SUMMARY OF THE INVENTION

It is an object of the invention to provide methods for screening foranti-cancer drugs.

It is another object of the invention to provide cell lines useful forscreening for anti-cancer drugs.

These and other objects of the invention are provided by one or more ofthe embodiments described below. In one embodiment of the invention, amethod for screening test compounds to identify those which arepotential anti-tumor agents is provided. The method comprises the stepsof: determining DNA content of checkpoint gene-defective human cellsincubated in the presence and in the absence of a test compound, whereina test compound which causes DNA accumulation in the checkpointgene-defective cell is identified as a potential anti-tumor agent.

In another embodiment of the invention, a different method of screeningfor potential anti-tumor agents is provided. The method comprises thesteps of: determining viability or apoptosis of checkpointgene-defective human cells incubated in the presence and in the absenceof a test compound; selecting a test compound which causes cell death orapoptosis in the checkpoint gene-defective cell.

In yet another embodiment of the invention a homozygous checkpointgene-defective human cell line is provided.

In still another embodiment of the invention a pair of isogenic celllines is provided. The first cell line is a homozygous checkpointgene-defective human cell line and the second cell line is a homozygouscheckpoint gene-normal human cell line.

These and other embodiments of the invention provide the art with newmethods and cell lines for screening potential anti-tumor agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows apoptosis in p21-deficient cells. p21-positive cells (1a,c,e) and p21-deficient cells (1 b,d,f) were grown for 90 hours in thepresence of adriamycin and viewed with phase contrast microscopy (1a,b), after staining with the DNA-binding dye H33258 (1 c,d), or afterstaining with the TUNEL assay to detect fragmented DNA (1 c,d).

FIG. 2 demonstrates the kinetics of apoptosis following treatment ofp21-deficient cells with drugs or irradiation. Cells were treated with(a) adriamycin (0.2 ug/ml), (b) etoposide (5 ug/ml), or (d) camptothecan(0.1 ug/ml), or (c) gamma irradiated (12 Gy). At the indicated times,cells were harvested, stained with the DNA-binding dye H33258, andviewed by fluorescence microscopy to determine the fraction of apoptoticcells.

FIG. 3 shows cell cycle analysis of drug treated cells. Cells with orwithout intact p21 genes were stained following various periods of drugtreatment and examined by flow cytometry. The cells were untreated (FIG.3 a, and 3 b), treated with adriamycin (FIG. 3 c, 3 d, 3 e, 3 f, 3 g,and 3 h) or etoposide (FIG. 3 m, 3 n, 3 o, and 3 p) for the indicatedtime periods, or treated with gamma irradiation and examined 30–60 hourslater (FIG. 3 i, 3 j, 3 k, and 3 l).

FIG. 4 demonstrates DNA synthesis and fluorescence in situ hybridizationin p21-deficient and p21-positive cells. Cells without (4 a, 4 b) orwith (4 c, 4 d) intact p21 genes were treated with adriamycin,pulse-labeled with BrdU to assess DNA synthesis, fixed and hybridizedwith a chromosome 3 probe by FISH (4 a, 4 c), then stained with theDNA-binding dye DAPI (4 a, 4 c) and anti-BrdU antibodies (4 b, 4 d).Hybridization signals from FISH are visualized as white dots, whilenuclear morphology is revealed by the blue DAPI stain. Note that asingle, lobulated nucleus is shown in 4 a and 4 b.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present inventors that checkpointgene-defective human cells are particularly useful for screeningpotential anti-tumor agents. This discovery is based on the observationthat agents which are already known in the art to be particularlyeffective anti-tumor agents have a pronounced and detectablephysiological effect on cells which are checkpoint gene-defective, e.g.,p21-negative. In the absence of a functional cell cycle checkpoint,DNA-damaged cells arrest in G2 but then undergo additional S phaseswithout intervening normal mitoses. They thereby acquire grosslydeformed, polyploid nuclei and subsequently die through apoptosis.

According to the present invention, potential therapeutic agents arescreened for the ability to cause DNA accumulation or cell death incheckpoint gene-defective human cells. Preferably, agents are screenedfor the ability to preferentially cause DNA accumulation or cell deathin checkpoint gene-defective human cells as compared to checkpointgene-normal human cells. More preferably, agents are screened for theability to cause DNA accumulation to at least four times the haploid DNAcontent of the checkpoint gene-defective human cells.

A checkpoint gene is a gene whose product is involved in regulation oftransitions between different cell cycle phases. Examples of suchtransitions or checkpoints are the initiation and the completion of DNAreplication (S phase) and of cell division (M phase). Severalcheckpoints are regulated by a family of protein kinases, the CDKs, andtheir obligate activating partners, the cyclins. Deregulation of atransition occurs as a consequence of either the aberrant expression ofpositive regulators, such as the cyclins, or the loss of negativeregulators, such as the CDK inhibitors, e.g., p21, p16, p15, p27 andp53. Any gene encoding positive or negative regulators of cell cyclecheckpoints is a checkpoint gene and is contemplated for use in thepresent invention. In particular, genes encoding CDK inhibitors, e.g.,p21, p16, p15, p27, and p53 are preferred. A checkpoint gene-defectivecell is one which lacks one or two wild-type checkpoint gene alleles oris deficient in a checkpoint gene function. The defects may be due toabnormal expression of a checkpoint gene or mutation in a checkpointgene. In a preferred embodiment, a checkpoint gene-defective cell lacksboth wild-type checkpoint gene alleles, i.e., is homozygous.

Any means known in the art to generate a cell line which is defective ina known checkpoint gene can be used to obtain the checkpointgene-defective cells. For example, a colonic cell line can be used togive rise to an isogenic p21-negative colonic cell line by promoterlesshomologous recombination⁵. The disclosure of reference 5 is expresslyincorporated herein. A cell with two wild-type alleles of a checkpointgene is a checkpoint gene-normal cell, for purposes of the presentinvention. Preferably, the checkpoint gene-defective cell used in theassay is the same type of cell (organ source) as the checkpointgene-defective cell. More preferably the two cell lines are isogenic.

The DNA content of a cell incubated in the presence or absence of a testcompound can be determined by any means known in the art. A DNA-bindingdye may be employed to measure the DNA content. Procedures are availablein the art to harvest cells, stain them with a DNA-binding dye, e.g.,propidium iodide or H33258, and measure the incorporation of theDNA-binding dye by flow cytometry. Flow cytometry provides an ordinaryartisan with information on the percentage of cells in a testedpopulation with a diploid DNA content (2C), four times of haploid (4C),eight times of haploid (8C), etc. Alternatively, the DNA content of acell can be determined by fluorescence in situ hybridization (FISH).Cells can be harvested, fixed on a slide, and hybridized with achromosome probe. The DNA probe can be labeled and detected underfluorescence microscopy by any means known in the art. In one particularmethod, the DNA probe is biotinylated by nick translation and detectedwith fluorescein isothiocyanate (FITC) conjugated to avidin. The DNAcontent of a cell can be obtained by quantifying the intensity of thefluorescein signal through digital image acquisition and processing,which are readily available in the art.

It is well known in the art that viability of a cell can be determinedby contacting the cell with a dye and viewing it under a microscope.Viable cells can be observed to have an intact membrane and do notstain, whereas dying or dead cells having “leaky” membranes do stain.Incorporation of the dye by the cell indicates the death of the cell.The most common dye used in the art for this purpose is trypan blue.Viability of cells can also be determined by detecting DNA synthesis.Cells can be cultured in cell medium with labeled nucleotides, e.g., ³Hthymidine. The uptake or incorporation of the labeled nucleotidesindicates DNA synthesis. In addition, colones formed by cells culturedin medium indicate cell growth and is another way to test viability ofthe cells.

Apoptosis is a specific mode of cell death recognized by acharacteristic pattern of morphological, biochemical, and molecularchanges. Cells going through apoptosis appear shrunken, and rounded;they also can be observed to become detached from culture dish. Themorphological changes involve a characteristic pattern of condensationof chromatin and cytoplasm which can be readily identified bymicroscopy. When stained with a DNA-binding dye, e.g., H33258, apoptoticcells display classic condensed and punctate nuclei instead ofhomogeneous and round nuclei.

A hallmark of apoptosis is endonucleolysis, a molecular change in whichnuclear DNA is initially degraded at the linker sections of nucleosomesto give rise to fragments equivalent to single and multiple nucleosomes.When these DNA fragments are subjected to gel electrophoresis, theyreveal a series of DNA bands which are positioned approximately equallydistant from each other on the gel. The size difference between the twobands next to each other is about the length of one nucleosome, i.e.,120 base pairs. This characteristic display of the DNA bands is called aDNA ladder and it indicates apoptosis of the cell. Apoptotic cells canbe identified by flow cytometric methods based on measurement ofcellular DNA content, increased sensitivity of DNA to denaturation, oraltered light scattering properties. These methods are well known in theart and are within the contemplation of the invention.

Abnormal DNA breaks are also characteristic of apoptosis and can bedetected by any means known in the art. In one preferred embodiment, DNAbreaks are labeled with biotinylated dUTP (b-dUTP). Cells are fixed andincubated in the presence of biotinylated dUTP with either exogenousterminal transferase (terminal DNA transferase assay; TdT assay) or DNApolymerase (nick translation assay; NT assay). The biotinylated dUTP isincorporated into the chromosome at the places where abnormal DNA breaksare repaired, and are detected with fluorescein conjugated to avidinunder fluorescence microscopy.

The following examples are provided for exemplification purposes onlyand are not intended to limit the scope of the invention which has beendescribed in broad terms above.

EXAMPLE 1 Apoptosis In p21-Deficient Cells

HCT116 cells with (+/+) or without (−/−) p21 genes were generated byhomologous recombination. Logarithmically-growing cells in McCoy's 5Amedium with 10% fetal calf serum were treated with Adriamycin (0.2ug/ml) for 90 hours. In FIG. 1 b, the adherent cells were harvested bytrypsinization and combined with cells floating in the media. Afterwashing with Hanks Buffered Saline (HBS; Life Technologies), cells wereresuspended in 40 ul HBS and added to 360 ul of a solution containing0.7% NP-40, 4.7% formaldehyde and 11 ug/ml H133258 in phosphate bufferedsaline (PBS). Cells were then viewed under UV excitation andphotographed with a Nikon Labophot microscope. In FIG. 1 c, adherentcells were fixed in 1% formaldehyde, then incubated in the presence ofexogenous terminal transferase and biotin-11-dUTP as previouslydescribed⁷. Labeled cells were detected by immunoperoxidase staining(Vector).

When treated with Adriamycin, the parental (p21^(+/+)) cells remainedattached to the plate and appeared morphologically normal, as do othercolorectal epithelial cell lines which growth arrest following DNAdamage³. In contrast, the p21-deficient cells (p21^(−/−)) shrank,rounded, and detached from the dish, suggesting apoptosis (FIG. 1 a and1 b). To confirm this suggestion, cells were stained with theDNA-binding dye H33258, revealing a classic condensed and punctatenuclear morphology in the p21^(−/−) cells, while the p21^(+/+) cellnuclei were homogeneous and round (FIG. 1 c and 1 d). The condensed,punctate nuclei were stained intensely by TUNEL^(6,7), which detects thepresence of DNA breaks characteristic of apoptosis (FIG. 1 e and 1 f).

EXAMPLE 2 Kinetics of Apoptosis Following the Treatment of p21-DeficientCells with Drugs or Irradiation

Cells were grown, fixed, and stained as described in Example 1.Irradiation was delivered at 1 Gy/minute using a ¹³⁷Cs source. Apoptoticcells were recognized as condensed, punctate nuclei (examples in FIG. 1d) or ghosts with faintly stained, degrading nuclei. At least 200 cellswere counted for each time point, and the experiment was repeated withresults virtually identical to those shown. The error bars represent thePoisson standard deviation determined by taking the square root ofcounted events and converting it to percent abundance. All counting wasdone in a blinded fashion. The (+/−) cells contained one normal p21allele and one allele deleted by homologous recombination⁵.

Time course analyses demonstrated that apoptosis was complete between 60and 90 hours following Adriamycin treatment (FIG. 2 a). The response toAdriamycin was found to be identical in a second, independently isolatedp21^(−/−) clone (FIG. 2 a).

To determine whether these observations were generalizable with respectto DNA-damaging agents, we treated the cells with the topoisomerase IIinhibitor etoposide, gamma irradiation, and the topoisomerase Iinhibitor camptothecan. After etoposide or irradiation, the parentalp21^(+/+) cells remained healthy while their p21^(−/−) derivatives died,as judged both by phase contrast microscopy and H33258 staining (FIG. 2b, 2 c, and 2 d). With camptothecan, the parental p21^(+/+) cellseventually underwent apoptosis, but this was significantly delayedcompared to p21-deficient cells (FIG. 2 d). Cells with one normal copyof p21 and one deleted copy (p21^(+/−)) behaved similarly to theparental cells after treatment with Adriamycin and etoposide (FIG. 2 a,and 2 b), while treatment with camptothecan or gamma irradiationrevealed a heterozygote effect (FIG. 2 c, and 2 d).

EXAMPLE 3 Cell Cycle Analysis of Drug-Treated Cells

Cell growth, drug treatment, fixation, and staining with H33258 wasperformed as described in Example 2. Flow cytometry was performed aspreviously described⁵.

Flow cytometry demonstrated that specific cell cycle changes wereassociated with this apoptotic process. Following 30 hours of exposureto Adriamycin, p21^(+/+) cells were blocked in G1 and G2 phases, withfew cells in S (FIG. 3 c). In contrast, no G1 block was evident in thep21^(−/−) cells⁵, so that a nearly pure population of G2-arrested cellswas observed (FIG. 3 d). With longer treatments, the flow cytometryprofile of p21^(+/+) cells remained largely unchanged (FIG. 3 e, and 3g), indicating a stable growth arrest⁸, while the p21^(−/−) cells beganto accumulate DNA in excess of 4C, then died (FIG. 3 f, and 3 h). Thesecharacteristic changes—abnormally high DNA content coupled withapoptosis—were observed in p21^(−/−) cells following treatment with eachof the DNA-damaging drugs (examples in FIG. 3 f, 3 h, 3 n, 3 p).Following gamma irradiation, there was a particularly strikingaccumulation of polyploid cells, with 33% and 15% of nuclei exhibitingDNA contents of 8C and 16C, respectively (FIG. 3 l).

EXAMPLE 4 DNA Synthesis and Fluorescence In Situ Hybridization inp21-Deficient Cells

Cells were grown and treated with Adriamycin as described in Example 1.After 60 hours of incubation, the cells were pulse-labeled with BrdU (5uM) for 1.5 hours. The cells were then harvested, fixed on glass slideswith methanol:acetic acid (3:1), treated with RNAse and pepsin²⁸, andFISH performed²⁹ with a P1 clone derived from 3p21.1-3. The P1 probe DNAwas biotinylated by nick translation and detected with fluoresceinisothiocyanate (FITC) conjugated to avidin³⁰. BrdU incorporation wasdetected by indirect immunofluorescence using an anti-BrdU antibody (5ug/ml, Pharmingen) and an anti-mouse IgG TRITC conjugate (Sigma). Cellswere counterstained with 0.1 ug/ml DAPI. Photographs were taken using aCCD camera (Photometrics) after digital image acquisition and processingas described previously²⁹

Previous studies have suggested that nuclei do not re-enter S phaseduring a block in G2 or M due either to the absence of required(positive) effectors or to the presence of negative effectors¹⁻³. Ourstudies clearly show that a major effector of this M/S coupling inHCT116 cells is negative and identify it as p21. The p21 protein couldachieve its effect by inhibiting cyclin-cdk complexes or by inhibitingproliferating cell nuclear antigen, a polymerase processivity factorrequired for DNA replication^(9,10). The lack of inhibition ofcyclin-cdk complexes could also promote the subsequent apoptosis¹¹⁻¹⁴.The effects of p21 deletion described here are in some ways differentfrom those caused by deletion of p21 in mouse fibroblasts^(15,16).Whether such differences reflect cell type or species-specific factorsis unclear; it is known that the control of S/M coupling isheterogeneous, differing significantly, for example, between differentmammalian cell types^(17,18) and between fission yeast and buddingyeast².

The data also demonstrate that the absence of p21 renders cellsremarkably sensitive to apoptosis following treatment with severalcancer therapeutics. The cellular events accompanying this sensitivityappear uniform-cells initiate and often complete entire rounds of DNAsynthesis in the absence of mitosis, leading to gross nuclearabnormalities followed by programmed cell death. These results provideexperimental evidence for the hypothesis that disruption of checkpointfunction could make mammalian cells more sensitive to chemotherapeuticagents^(1-3,18,19). They also have potential implications forunderstanding the successes and failures of current cancer therapy, asnaturally occurring cancers often have alterations of cyclins, cdk's, orcdk inhibitors (including p21) which could make them functionallyequivalent to p21^(−/−) cells²⁰⁻²². The data also suggest that thesensitivity of a cell to chemotherapeutic agents and irradiation willdepend on the relative intactness of both its M/S coupling and apoptoticcontrols^(17,23). This may explain why p53-deficient cells arerelatively resistant to cancer drugs²⁴, as their apoptotic response isabnormal²⁴⁻²⁷, unlike that in p21-deficient cells^(15,16).

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein, however, is notto be construed as limited to the particular forms disclosed, since theyare to be regarded as illustrative rather than restrictive. Variationsand changes may be made by those skilled in the art without departingfrom the spirit of the invention.

REFERENCES

-   1. Murray, A. W. Nature 359, 599–604 (1992).-   2. Nurse, P. Cell 79, 547–550 (1994).-   3. Hartwell, L. H. & Kastan, M. B. Science 266, 1821–1828 (1994).-   4. Nasmyth, K. & Hunt, T. Nature 366, 634–635 (1993).-   5. Waldman, T., Kinzler, K. W. & Vogelstein, B. Cancer Res. 55,    5187–5190 (1995).-   6. Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. J. Cell Biol. 119,    493–501 (1992).-   7. Gorczyca, W., Gong, J. & Darzynkiewicz, Z. Cancer Res. 53,    1945–1951 (1993).-   8. Di Leonardo, A. et al. Genes and Development 8, 2540–2551 (1994).-   9. Waga, S. Hannon, G. J., Beach, D. & Stillman, B. Nature 369,    574–578 (1994).-   10. Flores-Rozas, H. et al. Proc. Natn. Acad. Sci. U.S.A. 91,    8655–8699 (1994).-   11. Heald, R., McLoughlin, M. & McKeon, F. Cell 74, 463–474 (1993).-   12. Meikrantz, W., Gisselbrecht, S. , Tam, S. W., and Schlegel, R.    Proc. Natn. Acad. Sci. U.S.A. 91, 3754–3758 (1994).-   13. Hoang, A. T et al. Proc. Nat. Acad. Sci. U.S.A. 91, 6875–6879    (1994).-   14. Shi, L. et al. Science 263, 1143–1145 (1994).-   15. Deng, C. et al. Cell 82, 675–684 (1995).-   16. Brugarolas, J. et al. Nature 377, 552–557 (1995).-   17. Woods, C. M. et al. Molecular Medicine 1, 506–526 (1995).-   18. Kung, A. L., Sherwood, S. W., and Schimke, R. T. Proc. Natn.    Acad. Sci. U.S.A. 87: 9553–9557 (1990).-   19. Kung, A. L., Zetterberg, A., Sherwood, S. W. & Schimke, R. T.    Cancer Res. 50, 7307–7317 (1990).-   20. Gao, X. et al. Oncogene 11, 1395–1398 (1995).-   21. Hunter, T. & Pines, J. Cell 79, 573–582 (1994).-   22. Sherr, C. J. & Roberts, J. M. Genes and Development 9: 1149–1163    (1995).-   23. Cross, S. M. et al. Science 267, 1353–1356 (1995).-   24. Lowe, S. W. et al. Cell 74, 957–967 (1993).-   25. Yonish-Rouach, E. et al. Nature 352, 345–7 (1991).-   26. Lowe, S. W. et al. Nature 362, 847–849 (1993).-   27. Clarke, A. R. et al. Nature 362, 849–852 (1993).-   28. Ried, T. et al. Genes, Chromosomes, & Cancer 4, 69–74 (1992).-   29. Lengauer, C. et al. Genetic Analysis Techniques and Applications    11, 140–147 (1994).-   30. Lichter, P. & Cremer, T. Human Cytogenetics: A Practical    Approach, 157–192 (Oxford, IRL University Press, 1992).

1. A method of screening for potential anti-tumor agents, comprising thesteps of: determining apoptosis of homozygous checkpoint gene-defectivehuman cells incubated in the presence and in the absence of a testcompound, wherein the checkpoint gene encodes a negative regulator ofcell cycle checkpoint selected from the group consisting of p21, p16,p15, p27, and p53; selecting a test compound which causes apoptosis inthe checkpoint defective cells.
 2. The method of claim 1 wherein theapoptosis of the cells is determined by staining the cells with aDNA-binding dye and observing chromosomes of the cells, condensation ofthe chromosomes indicating apoptosis of the cells.
 3. The method ofclaim 2 wherein the DNA-binding dye is H33258.
 4. The method of claim 1wherein the apoptosis of the cells is determined by subjecting DNA ofthe cell to gel electrophoresis, wherein observation of a DNA ladderindicates apoptosis of the cells.
 5. The method of claim 1 wherein theapoptosis of the cells is determined by detecting abnormal breaks in DNAof the cells, wherein abnormal breaks in the DNA indicate apoptosis ofthe cells.
 6. The method of claim 5 wherein the abnormal breaks in theDNA are detected by terminal DNA transferase assay.
 7. The method ofclaim 5 wherein the abnormal breaks in the DNA are detected by nicktranslation assay.
 8. The method of claim 1 wherein the checkpoint geneis p53.
 9. The method of claim 8 wherein the checkpoint gene-defectivehuman cells are colonic cells.
 10. The method of claim 1 furthercomprising the steps of: determining apoptosis of checkpoint gene-normalhuman cells incubated in the presence and in the absence of the selectedtest compound; identifying a selected test compound which preferentiallycauses apoptosis in the checkpoint gene-defective cells as compared tothe checkpoint gene-normal cells.
 11. The method of claim 10 wherein theapoptosis of the cells is determined by staining the cells with aDNA-binding dye and observing chromosomes of the cells, condensation ofthe chromosomes indicating apoptosis of the cells.
 12. The method ofclaim 11 wherein the DNA-binding dye is H33258.
 13. The method of claim10 wherein the apoptosis of the cells is determined by subjecting DNA ofthe cell to gel electrophoresis, wherein observation of a DNA ladderindicates apoptosis of the cells.
 14. The method of claim 10 wherein theapoptosis of the cells is determined by detecting abnormal breaks in DNAof the cells, wherein abnormal breaks in the DNA indicate apoptosis ofthe cells.
 15. The method of claim 14 wherein the abnormal breaks in theDNA are detected by terminal DNA transferase assay.
 16. The method ofclaim 14 wherein the abnormal breaks in the DNA are detected by nicktranslation assay.
 17. The method of claim 10 wherein the checkpointgene is p53.
 18. The method of claim 17 wherein the checkpointgene-defective human cells are colonic cells.
 19. A method of screeningfor potential anti-tumor agents, comprising the steps of: determiningapoptosis of homozygous checkpoint gene-defective human cells incubatedin the presence and in the absence of a test compound, wherein thecheckpoint gene is p21; determining apoptosis of checkpoint gene-normalhuman cells incubated in the presence and in the absence of the selectedtest compound; identifying a selected test compound which preferentiallycauses apoptosis in the checkpoint gene-defective cells as compared tothe checkpoint gene-normal cells.
 20. The method of claim 10 wherein thecheckpoint gene-normal human cells and the checkpoint gene-defectivehuman cells are isogenic.
 21. The method of claim 19 wherein thecheckpoint gene-normal human cells and the checkpoint gene-defectivehuman cells are isogenic.